Modulation of higher order chromatin conformation in mammalian cell nuclei can be mediated by polyamines and divalent cations

The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisatio

ATP depletion leads to increased chromatin compaction.

<p>(A) FLIM images show the reduction of fluorescence lifetime in Hela^H2B-2FP cells upon ATP depletion and an increase when the drugs are washed away. The increase in compaction is also seen in the intensity images (Scale bar –10 µm). (B) The chart shows the average decrease and subsequent increase in EGFP fluorescence lifetime values normalised to the lifetime of the control nuclei. Error bars indicate standard deviation for values collected for 13 cells from 3 independent experiments. (* p-value = 3.7×10⁻⁸,+p-value = 0.0046).</p

Mitotic chromatin compacts with ATP depletion.

<p>(A) FLIM images showing a decrease in EGFP fluorescence lifetime on ATP depletion and its subsequent increase to control levels when the drugs are washed away in Hela^H2B-2FP cells (Scale bar - 10 µm). (B) Chart showing the average EGFP fluorescence lifetime normalised to control values from 9 mitotic cells acquired from 7 individual experiments showing changes upon ATP depletion and repletion. Error bars indicate standard deviation. (* p-value = 3.7×10⁻⁶,+p-value = 0.144).</p

ATP depletion leads to an increase in intracellular free calcium levels.

<p>Fluo4-AM loaded HeLa cells were imaged before and after ATP depletion. The increase in fluorescence indicates an increase in free intracellular calcium levels (p-value = 0.0001). The increase in chromatin compaction (yellow arrow head) can be seen from Hoechst staining after ATP depletion. (Scale bar –10 µm).</p

Increase in polyamine concentration leads to increased chromatin compaction in permeabilised cells.

<p>(A) FLIM images of permeabilised Hela^H2B-2FP cells with increasing Spermidine³⁺ concentration showing a reduction in EGFP fluorescence lifetime. The increase in compaction is also seen with the fluorescence intensity image. (Scale bar –10 µm) (B) FLIM images of permeabilised Hela^H2B-2FP cells with increasing Spermine⁴⁺ concentration showing a reduction in the calculated EGFP fluorescence lifetime. The increase in compaction is also seen with the fluorescence intensity image. (Scale bar –10 µm) (C) The average fluorescence lifetime normalised to control values of the Hela^H2B-2FP cells are plotted against the Spermidine³⁺ concentration. Error bars indicate standard deviation for values calculated for 15 cells from 3 independent experiments. (D) The average fluorescence lifetime normalised to control values for each the cells are plotted against the Spermine⁴⁺ concentration. Error bars indicate standard deviation for values calculated for 22 cells from 3 independent experiments.</p

Increasing calcium concentration in live cells leads to an increase in chromatin compaction.

<p>(A) Fluo4 fluorescence images showing an increase in free intracellular calcium levels on treating the cells with Ca²⁺ ionophore. (B) FLIM images showing a decrease in fluorescence lifetime of Hela^H2B-2FP cells that have been treated with Ca²⁺ ionophore. (C) Chart showing the mean lifetime values of individual Hela^H2B-2FP cells before (blue) and after treating with Ca²⁺ ionophore. (Scale bar –10 µm).</p

Electron spectroscopic imaging of permeabilised cells showing increased chromatin compaction with increase in Ca²⁺ concentration.

<p>(A) Electron spectroscopic imaging showing chromatin in yellow and non chromosomal proteins in blue of permeabilised HeLa cells containing varying levels of Mg²⁺ ions. (B) ESI combined with tomography showing the three dimensional structure of the chromatin of permeabilised HeLa cells containing varying levels of Mg²⁺ ions.</p

Increasing divalent cation concentration leads to an increase in chromatin compaction in permeabilised cells.

<p>(A) FLIM images of permeabilised Hela^H2B-2FP cells with increasing Ca²⁺ concentration. There is a decrease in lifetime with increasing Ca²⁺ concentrations and an increase with the addition of 12 mM of EDTA. The increase in compaction is also seen with the fluorescence intensity image. (Scale bar –10 µm). (B) Average EGFP fluorescence lifetime normalised to the value of control cells are plotted against the varying concentration of Ca²⁺ and Mg²⁺. Error bars indicate standard deviation calculated for 16 and 15 cells respectively from 4 independent experiments.</p