Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging.
Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.
Weidemann
Thomas
T
Deutsches Krebsforschungszentrum, Division Biophysics of Macromolecules, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.
Wachsmuth
Malte
M
Knoch
Tobias A
TA
Müller
Gabriele
G
Waldeck
Waldemar
W
Langowski
Jörg
J
D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D009707:Nucleosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D009707:Nucleosomes