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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 depletes intracellular ATP levels, inhibits transcription, induces relocation of the cellular polyamine pool to the nucleus and reduces the staining density of histone H3 with antibody. a The intracellular concentration of ATP in untreated, OND-exposed and recovering cells was determined using a luciferase dependent assay. b Global rates of transcription, determined by the incorporation of bromouridine into RNA, in untreated cells, cells under OND and cells recovering from OND are presented. HL-1 cells, either untreated or subjected to 1 hour of OND, were fixed, permeabilized and stained either with anti-polyamine antibody (c, d) or with anti-total H3 antibody (e, f) and counterstained with the fluorescent DNA binding dye Hoechst 33342. Cells were then examined using confocal microscopy. The content of immunostained histone H3 was evaluated by SMLM in untreated (e) and OND-treated (f) HL-1 cells. BrU bromouridine. The error bars represent the standard deviation of three independent samples
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Fig6: OND depletes intracellular ATP levels, inhibits transcription, induces relocation of the cellular polyamine pool to the nucleus and reduces the staining density of histone H3 with antibody. a The intracellular concentration of ATP in untreated, OND-exposed and recovering cells was determined using a luciferase dependent assay. b Global rates of transcription, determined by the incorporation of bromouridine into RNA, in untreated cells, cells under OND and cells recovering from OND are presented. HL-1 cells, either untreated or subjected to 1 hour of OND, were fixed, permeabilized and stained either with anti-polyamine antibody (c, d) or with anti-total H3 antibody (e, f) and counterstained with the fluorescent DNA binding dye Hoechst 33342. Cells were then examined using confocal microscopy. The content of immunostained histone H3 was evaluated by SMLM in untreated (e) and OND-treated (f) HL-1 cells. BrU bromouridine. The error bars represent the standard deviation of three independent samples

Mentions: We then postulated that chromatin compaction induced by OND is consequent upon ATP depletion. Under normal conditions, divalent cations and polyamines associate with the polyphosphate group of ATP. However, if ATP levels are reduced, these may, by mass-action, relocate to the sugar-phosphate backbone of nucleic acid, thereby promoting chromatin compaction through effecting charge shielding. OND reduces intracellular ATP levels by 90 %, which recovers upon cessation of OND with kinetics similar to that of chromatin relaxation (Fig. 6a). Furthermore, OND promotes a global decrease of transcription by approximately 90 %, as estimated by mass spectrometric determination of bromouridine incorporation into nascent RNA (Fig. 6b). We then described the distribution of the intracellular polyamine pool using immunocytochemistry. Anti-polyamine staining of untreated HL-1 cells results in a punctate, predominantly cytoplasmic distribution with a low level of intranuclear staining (Fig. 6c). This most likely reflects ATP-rich mitochondria present in the cytoplasm of cardiomyocytes. In contrast, OND treatment for 1 hour results in the transfer of a significant part of the cellular polyamine pool to the nucleus (Fig. 6d) with particularly intense staining of RNA rich nucleoli. Additionally, SMLM of histone H3 indicates that, in comparison with untreated cells (Fig. 6e), OND treatment (Fig. 6f) reduces the apparent density of chromatin-associated histone H3 in the nucleus from 3813 ± 250 per μm2 to 842 ± 503 per μm2, whereas levels observed in the cytoplasm remain similar at 250 per μm2. Furthermore, the localization density achieved for total H3 is much lower than for DNA binding dyes, and is insufficient to discern OND-induced chromatin compaction.Fig. 6


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 depletes intracellular ATP levels, inhibits transcription, induces relocation of the cellular polyamine pool to the nucleus and reduces the staining density of histone H3 with antibody. a The intracellular concentration of ATP in untreated, OND-exposed and recovering cells was determined using a luciferase dependent assay. b Global rates of transcription, determined by the incorporation of bromouridine into RNA, in untreated cells, cells under OND and cells recovering from OND are presented. HL-1 cells, either untreated or subjected to 1 hour of OND, were fixed, permeabilized and stained either with anti-polyamine antibody (c, d) or with anti-total H3 antibody (e, f) and counterstained with the fluorescent DNA binding dye Hoechst 33342. Cells were then examined using confocal microscopy. The content of immunostained histone H3 was evaluated by SMLM in untreated (e) and OND-treated (f) HL-1 cells. BrU bromouridine. The error bars represent the standard deviation of three independent samples
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: OND depletes intracellular ATP levels, inhibits transcription, induces relocation of the cellular polyamine pool to the nucleus and reduces the staining density of histone H3 with antibody. a The intracellular concentration of ATP in untreated, OND-exposed and recovering cells was determined using a luciferase dependent assay. b Global rates of transcription, determined by the incorporation of bromouridine into RNA, in untreated cells, cells under OND and cells recovering from OND are presented. HL-1 cells, either untreated or subjected to 1 hour of OND, were fixed, permeabilized and stained either with anti-polyamine antibody (c, d) or with anti-total H3 antibody (e, f) and counterstained with the fluorescent DNA binding dye Hoechst 33342. Cells were then examined using confocal microscopy. The content of immunostained histone H3 was evaluated by SMLM in untreated (e) and OND-treated (f) HL-1 cells. BrU bromouridine. The error bars represent the standard deviation of three independent samples
Mentions: We then postulated that chromatin compaction induced by OND is consequent upon ATP depletion. Under normal conditions, divalent cations and polyamines associate with the polyphosphate group of ATP. However, if ATP levels are reduced, these may, by mass-action, relocate to the sugar-phosphate backbone of nucleic acid, thereby promoting chromatin compaction through effecting charge shielding. OND reduces intracellular ATP levels by 90 %, which recovers upon cessation of OND with kinetics similar to that of chromatin relaxation (Fig. 6a). Furthermore, OND promotes a global decrease of transcription by approximately 90 %, as estimated by mass spectrometric determination of bromouridine incorporation into nascent RNA (Fig. 6b). We then described the distribution of the intracellular polyamine pool using immunocytochemistry. Anti-polyamine staining of untreated HL-1 cells results in a punctate, predominantly cytoplasmic distribution with a low level of intranuclear staining (Fig. 6c). This most likely reflects ATP-rich mitochondria present in the cytoplasm of cardiomyocytes. In contrast, OND treatment for 1 hour results in the transfer of a significant part of the cellular polyamine pool to the nucleus (Fig. 6d) with particularly intense staining of RNA rich nucleoli. Additionally, SMLM of histone H3 indicates that, in comparison with untreated cells (Fig. 6e), OND treatment (Fig. 6f) reduces the apparent density of chromatin-associated histone H3 in the nucleus from 3813 ± 250 per μm2 to 842 ± 503 per μm2, whereas levels observed in the cytoplasm remain similar at 250 per μm2. Furthermore, the localization density achieved for total H3 is much lower than for DNA binding dyes, and is insufficient to discern OND-induced chromatin compaction.Fig. 6

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