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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 does not induce core histone displacement from chromatin but does decrease the mobility of the linker histone H1. We first demonstrated that HeLa cells stably transfected with either histone H2B-mCherry or histone H1.1-green fluorescent protein (GFP) respond to 1 hour of OND by undergoing chromatin compaction. a Comparison of untreated (UT) cells (top panels) with cells exposed to 1 hour of OND (bottom panels) by confocal microscopy clearly indicates that chromatin of HeLa cells compacts upon OND treatment. b We then evaluated the mobility of the core histone H2B using FRAP on untreated (upper panel) and on OND-treated (lower panel) cells. The recovery after photobleaching was extremely slow for both conditions, indicating that OND does not induce displacement of H2B from chromatin. c We then evaluated the mobility of the linker histone H1 in untreated and OND-treated HeLa cells. As previously reported [49, 50], histone H1 is mobile, and is somewhat less mobile in heterochromatin than in euchromatin. d OND-induced chromatin compaction dramatically reduces the mobility of histone H1, indicating that the extent of chromatin compaction in OND is considerably higher than that between euchromatin and heterochromatin
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Fig7: OND does not induce core histone displacement from chromatin but does decrease the mobility of the linker histone H1. We first demonstrated that HeLa cells stably transfected with either histone H2B-mCherry or histone H1.1-green fluorescent protein (GFP) respond to 1 hour of OND by undergoing chromatin compaction. a Comparison of untreated (UT) cells (top panels) with cells exposed to 1 hour of OND (bottom panels) by confocal microscopy clearly indicates that chromatin of HeLa cells compacts upon OND treatment. b We then evaluated the mobility of the core histone H2B using FRAP on untreated (upper panel) and on OND-treated (lower panel) cells. The recovery after photobleaching was extremely slow for both conditions, indicating that OND does not induce displacement of H2B from chromatin. c We then evaluated the mobility of the linker histone H1 in untreated and OND-treated HeLa cells. As previously reported [49, 50], histone H1 is mobile, and is somewhat less mobile in heterochromatin than in euchromatin. d OND-induced chromatin compaction dramatically reduces the mobility of histone H1, indicating that the extent of chromatin compaction in OND is considerably higher than that between euchromatin and heterochromatin

Mentions: We wished to discriminate between possible explanations underlying the approximately 80 % reduction in histone H3 staining upon OND treatment, as determined by SMLM. Potentially, this observation could arise through compaction restricting the accessibility of antibody to chromatin and/or through the direct loss of core histones from chromatin. By inference, histone loss from chromatin would liberate a highly mobile pool of histones, in contrast to their limited mobility when present in chromatin. We therefore used FRAP on live cells to estimate the mobility of histone H2B labeled with mCherry in untreated HeLa cells and HeLa cells in an ischemic environment. We selected H2B, which along with H2A, and in contrast to H3 and H4, exhibits significant exchange [48]. Consequently, FRAP analysis of H2B-mCherry is an appropriate marker for estimating OND-induced displacement of core histones. As shown in Fig. 7a, HeLa cells undergo chromatin compaction when subjected to 1 hour of OND, and significantly, H2B-mCherry retains a structured nuclear distribution, suggestive of chromatin compaction, indicating that a widespread release of core nucleosomes from chromatin does not occur upon OND treatment. FRAP measurements (Fig. 7b) of the mobility of H2B-mCherry confirm that OND does not increase the mobility of this core histone.Fig. 7


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 does not induce core histone displacement from chromatin but does decrease the mobility of the linker histone H1. We first demonstrated that HeLa cells stably transfected with either histone H2B-mCherry or histone H1.1-green fluorescent protein (GFP) respond to 1 hour of OND by undergoing chromatin compaction. a Comparison of untreated (UT) cells (top panels) with cells exposed to 1 hour of OND (bottom panels) by confocal microscopy clearly indicates that chromatin of HeLa cells compacts upon OND treatment. b We then evaluated the mobility of the core histone H2B using FRAP on untreated (upper panel) and on OND-treated (lower panel) cells. The recovery after photobleaching was extremely slow for both conditions, indicating that OND does not induce displacement of H2B from chromatin. c We then evaluated the mobility of the linker histone H1 in untreated and OND-treated HeLa cells. As previously reported [49, 50], histone H1 is mobile, and is somewhat less mobile in heterochromatin than in euchromatin. d OND-induced chromatin compaction dramatically reduces the mobility of histone H1, indicating that the extent of chromatin compaction in OND is considerably higher than that between euchromatin and heterochromatin
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: OND does not induce core histone displacement from chromatin but does decrease the mobility of the linker histone H1. We first demonstrated that HeLa cells stably transfected with either histone H2B-mCherry or histone H1.1-green fluorescent protein (GFP) respond to 1 hour of OND by undergoing chromatin compaction. a Comparison of untreated (UT) cells (top panels) with cells exposed to 1 hour of OND (bottom panels) by confocal microscopy clearly indicates that chromatin of HeLa cells compacts upon OND treatment. b We then evaluated the mobility of the core histone H2B using FRAP on untreated (upper panel) and on OND-treated (lower panel) cells. The recovery after photobleaching was extremely slow for both conditions, indicating that OND does not induce displacement of H2B from chromatin. c We then evaluated the mobility of the linker histone H1 in untreated and OND-treated HeLa cells. As previously reported [49, 50], histone H1 is mobile, and is somewhat less mobile in heterochromatin than in euchromatin. d OND-induced chromatin compaction dramatically reduces the mobility of histone H1, indicating that the extent of chromatin compaction in OND is considerably higher than that between euchromatin and heterochromatin
Mentions: We wished to discriminate between possible explanations underlying the approximately 80 % reduction in histone H3 staining upon OND treatment, as determined by SMLM. Potentially, this observation could arise through compaction restricting the accessibility of antibody to chromatin and/or through the direct loss of core histones from chromatin. By inference, histone loss from chromatin would liberate a highly mobile pool of histones, in contrast to their limited mobility when present in chromatin. We therefore used FRAP on live cells to estimate the mobility of histone H2B labeled with mCherry in untreated HeLa cells and HeLa cells in an ischemic environment. We selected H2B, which along with H2A, and in contrast to H3 and H4, exhibits significant exchange [48]. Consequently, FRAP analysis of H2B-mCherry is an appropriate marker for estimating OND-induced displacement of core histones. As shown in Fig. 7a, HeLa cells undergo chromatin compaction when subjected to 1 hour of OND, and significantly, H2B-mCherry retains a structured nuclear distribution, suggestive of chromatin compaction, indicating that a widespread release of core nucleosomes from chromatin does not occur upon OND treatment. FRAP measurements (Fig. 7b) of the mobility of H2B-mCherry confirm that OND does not increase the mobility of this core histone.Fig. 7

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