Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging

doi:10.1016/j.jmb.2003.08.063

J. Mol. Biol. (2003) 334, 229–240

Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging Thomas Weidemann, Malte Wachsmuth, Tobias A. Knoch Gabriele Mu¨ller, Waldemar Waldeck and Jo¨rg Langowski* Deutsches Krebsforschungszentrum Division Biophysics of Macromolecules, Im Neuenheimer Feld 580 D-69120 Heidelberg, Germany

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 H2BEYFP 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 mM locally, with mean values of 140(^ 28) mM, suggesting that a condensationcontrolled regulation of site accessibility takes place at length scales well below 200 nm. q 2003 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: chromatin; confocal microscopy; fluorescence correlation spectroscopy; histone metabolism; nuclear architecture

Introduction In eukaryotes, chromatin exists in multiple levels of organisation and compaction.1,2 The lowest and best-characterised level of packaging is the nucleosome, where 146 bp of DNA are wrapped helically around a cylindrical octamer core consisting of two copies of each histone class H2A, H2B, H3, and H4. Nucleosomes are maintained at regular Present addresses: T. Weidemann, Novartis Forschungsinstitut, Brunner Str. 59, A-1235 Wien, Austria; M. Wachsmuth and T. A. Knoch, Kirchhoff Institute for Physics, Molecular Biophysics, Ruprecht Karls University, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany. Abbreviations used: CLSM, confocal laser scanning microscopy; FCS, fluorescence correlation spectroscopy; MNase, micrococcal nuclease; CP, continuous photolabelling. E-mail address of the corresponding author: [email protected]

genomic distances of about 200 bp. Interactions between adjacent nucleosomes generate a filamentous higher order structure usually referred to as the 30 nm fibre. Although its precise conformation and folding mechanism are still under debate,3,4 an additional linear five- to sixfold condensation as compared to the “beads on the string” is widely assumed, and the fibre serves as the working model for a functional state of chromatin in interphase nuclei. Chromatin has been imaged in situ by selective staining of DNA with fluorescent dyes (e.g. ethidium bromide, Hoechst 33342, DAPI) or by fluorescent immunostaining of, e.g. histones in fixed and permeabilised cells. In recent years, much progress has been made in selective chromatin visualisation and DNA staining has been extended to in vivo applications.5 Microscopic methods have improved as well: confocal laser scanning microscopy (CLSM) provides a three-dimensional spatial resolution near the diffraction limit. However, in order to quantify DNA or proteins in

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

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vivo and to obtain absolute concentrations, two calibration steps are necessary: first, to determine the fluorescence yield of single dye molecules for imaging, and second, to relate the concentrations of bound dyes to the amount of labelled substrate. The latter is in particular difficult when the staining is reversible as in the case of intercalators. An alternative method of chromatin labelling was introduced by Kanda et al.6 They transfected a gene encoding one of the core histones fused with green fluorescent protein (H2B-GFP) into human HeLa cells. The tagged histones were stably incorporated into functional chromatin leading to fluorescent chromosomes visible throughout the cell cycle. Recent work has provided evidence for the functionality of these GFP-tagged histones. Photobleaching techniques like fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) have been successfully applied to measure the exchange kinetics of linker histone H1 and other nuclear components between different regions of the nucleus in vivo.7 – 9 In these studies, nuclei containing GFP-tagged H2B have served as a control for a fully immobilised nuclear compound. The most recent study by Kimura & Cook10 addressed the exchange kinetics of core histones with FRAP in more detail: except for a very small fast recovering fraction, they have observed exchange rates of H2B-GFP of the order of several hours. DNA counterstaining proved that the fluorescence distribution of core histones maps the DNA distribution, making such cell lines suitable to study nuclear structure11 – 13 and supplying useful reference for the relative localisation of other nuclear components. Nevertheless, a quantitative interpretation of such intensity variations is still missing. FCS14 is a method to measure mobility and absolute concentrations of fluorescent molecules in solution. This is achieved by analysing the fluorescence intensity fluctuations from a microscopic illuminated volume (of the order of a femtoliter and smaller) containing only a few fluorochrome molecules. The Brownian motion of the molecules in the observation volume, as well as the photophysics of the fluorophore, lead to fluorescence fluctuations that are analysed by time correlation functions, allowing to assess the hydrodynamic and photophysical properties of the molecules. Today, FCS is generally performed using confocal microscope optics,15,16 which allows probing cellular microenvironments that influence the diffusion and intramolecular dynamics with high spatial resolution. This makes FCS an intrinsically calibrated, non-invasive sensing tool for intracellular properties. In order to quantify fluorescence intensities in vivo and correlate them with mobility and absolute concentrations of the fluorophores at high spatial resolution and positioning accuracy, we have combined an FCS device with a confocal scanner into a system, which we have called fluorescence fluctuation microscope, FFM.17 The instrument

Counting Nucleosomes in Living Cells

allows confocal imaging and FCS measurements through the same optics with high positioning accuracy in cells. We studied a HeLa clone constitutively expressing H2B fused to Enhanced Yellow Fluorescent Protein (H2B-EYFP). With gel electrophoresis and FCS in vitro, the incorporation of fluorescent H2B into the chromatin fibre was determined quantitatively. Confocal images of the fluorescent nuclei were taken and calibrated with the fluorescence yield of single H2B-EYFP molecules derived from FCS in the cytoplasm. A background of mobile histones in the nucleoplasm was assessed with continuous photobleaching (CP). On the basis of these results, confocal images were transformed into density maps representing absolute nucleosome concentrations at each position of a nuclear cross-section. Since this combination of quantitative confocal imaging with FFM can be applied to many biological systems it may offer great potential to draw a quantitative picture of large scale molecular structures in living cells.

Results H2B-EYFP transfected cells show normal growth behaviour DNA encoding for human H2B (13.9 kDa) was inserted upstream of EYFP (27 kDa) in a mammalian expression vector. The chimerical gene was stably incorporated into the genome of a HeLa cell line by transfection and selection of several clones of stably reproducing fluorescent cells. Quantitative DNA precipitation of a well-defined number of cells yielded a DNA content of 6.7 £ 109 bp (4n) per cell in agreement with a tetraploid genome of clone Y. We chose a monoclonal cell line with fluorescent nuclei of moderate brightness for further investigations, which we denote in the following as clone Y. Since the EYFP fusion might cause some changes in chromatin maintenance during the cell cycle, we tested characteristics of cell cycle progression. FACS-analysis (fluorescence activated cell sorting) with DAPI-stained ethanolfixed cells showed that within the population of clone Y, about 58% of the cells are in G1/G0, 25% in S, and 15% in G2/M phase. The values (Table 1) are in good agreement with a non-fluorescent control C of HeLa cells. No significant variations were found in the cell division rates within experimental error. Nucleosomes of clone Y are spaced with an increased repeat length Micrococcal nuclease (MNase) digestion of isolated fluorescent nuclei leads to a typical nucleosomal ladder, which we fractionated on a sucrose gradient (Figure 1(a)). Oligonucleosomes could be resolved up to heptamers ðN ¼ 7Þ: Linear regression of the multiple-sized bands (Figure 1(b)) yielded a repeat length of 204(^ 3) bp for

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Table 1. Cell cycle progression HeLa cell line C Y

FACS fluorescence (a.u.) 1 34

a

Cell cyclea

Proliferation division time (hours) 18.2 16.9

G1/G0 (%)

S (%)

G2/M (%)

55 58

30 25

13 15

, 2% of the cells were found apoptotic in both cell lines.

clone Y, which is a significant increase compared to control HeLa cells C with 185(^ 10) bp in agreement with published results.18,19 Gel electrophoresis: about 5% of assembled H2B are fused to EYFP The clones Y and C were compared with respect to protein composition. Proteins of isolated nuclei were separated under denaturing conditions (SDS-PAGE) and visualised by Coomassie staining (Figure 2(a), left panel). The core histones appear as dominant bands in equal stoichiometrical ratios. No difference was found in the relative histone

Figure 1. Isolation of differently sized oligonucleosomes. (a) A 1% agarose gel of isolated DNA fragments obtained by MNase digestion of the nuclear chromatin of clone Y followed by fractionation on a sucrose gradient (10% to 30%). Oligonucleosomes up to heptamers ðN ¼ 1 – 7Þ were pooled for further usage (brackets). (b) The mean band sizes with respect to the reference (lane M in A) are plotted in dependence of the number of nucleosomes N located on the particular DNA fragment. The data were fitted by linear regression for clone Y (closed circles) and the non-fluorescent control C (open circles).

fraction between the transfected (lane Y) and nonfluorescent cells (lane C). Visualisation of H2B-EYFP fusion protein by a Western blot to the fluorescent domain of the construct resulted in a single band in the expected region slightly below 50 kDa (Figure 2(a), right panel). The detected position led to the identification of the fused peptide, the double band in Figure 2(b) is a result of slight proteolysis during purification, representing H2B-EYFP of purified oligonucleosomes.

Figure 2. Protein composition. (a) SDS-PAGE of whole nuclei followed by Coomassie staining. Beside various non-histone chromosomal proteins (NHCPs), the linker histone H1 and the core histones H3, H2B, H2A, and H4 are present as dominant bands (left panel; Y: transfected clone; C: non-fluorescent control; M: molecular weight marker). The H2B-EYFP was immunoblotted with an antibody against the fluorophore EYFP (right panel). (b) Quantitative SDS-PAGE by silver staining shows H2B-EYFP in fluorescent oligonucleosomes equivalent to 10 mg nucleosomal DNA (left panel). Band intensities were calibrated with a linearly titrated mass of recombinant H2A (right panel; recH2A).

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For quantitative determination of the protein concentrations, we used silver-stained SDS-PAGE gels that were scanned and quantitated. In order to avoid error due to non-linear concentration dependence of staining we compared the protein bands with a spectroscopically defined amount of recombinant H2A, which served as a calibration for the imaged grey values (Figure 2(b), graph). Oligonucleosomes corresponding to 10 mg of nucleosomal DNA carry 310(^ 40) ng of H2BEYFP, thus about 5% of the total numbers of H2B assembled into nucleosomes are genetically fused to EYFP. FCS: H2B-tagged nucleosome chains diffuse freely in solution For characterising EYFP-tagged oligonucleosomes in solution by FCS, we first studied spectroscopically the fluorescence properties of isolated tagged nucleosome chains from clone Y in their native state. Fractions from the sucrose gradient were pooled as indicated in Figure 1(a) (brackets) and transferred into aqueous buffer at low ionic strength and physiological pH. Absorption and fluorescence spectra as well as FCS measurements were collected from the same solutions. The decay of the FCS autocorrelation function contains information about the mobility and photophysics of the fluorophore, while its amplitude Gð0Þ is inversely proportional to the fluorophore concentration in the observation volume.20 Independently of size, we obtained emission spectra as reported for EYFP, and no spectral variations appeared for larger complexes up to heptamers (data not shown). The diffusion coefficient D of the oligonucleosomes was obtained from the decay time of the autocorrelation curves measured in FCS (e.g. Figure 3), which in addition to the diffusional correlation time contained two correlation times of , 30 ms and , 300 ms corresponding to the molecular “blinking” typical of autofluorescent proteins.21 Diffusion coefficients of differently sized complexes free in solution decrease from , 20 mm2/s for mononucleosomes below 10 mm2/s for heptamers and larger nucleosome chains, respectively (Figure 3(b)). These values are comparable to those observed with other methods, e.g. dynamic light scattering.22 The data prove that the isolated chromatin fragments exist in solution as dispersed particles, and measured quantities are not biased by non-specific aggregation, which is an important prerequisite for evaluation of the FCS amplitudes. FCS: 5% of incorporated H2B carry a fluorescent tag The linear relationship between the FCS autocorrelation amplitude Gð0Þ and the inverse of the average number of detectable particles in the observation volume had been experimentally verified for more than four orders of magnitude in

Figure 3. Fluorescent oligonucleosomes characterized in vitro. (a) Autocorrelation curves and absorption spectra (inset) of the same sample of freely diffusing fluorescent mononucleosomes (continuous) and hexamers (dotted) in TE, 20 mM KCl. cEYFP and cnuc indicate the concentrations of fluorescent H2B-EYFP and nucleosomes, respectively. (b) Diffusion coefficients D of various sized EYFP-tagged nucleosomes (for DNA compositions see Figure 1(a)). (c) Incorporation probability p for H2B-EYFP derived from FCS, absorption and gels for differently sized oligonucleosomes and mixtures thereof ðN . 5Þ: Two independent nucleosome preparations and several sets of FCS measurements have been averaged.

our setup.23 As an example, Figure 3(a) illustrates FCS measurements of mononucleosomes in comparison to hexamers. The lower amplitude for mononucleosomes corresponds to a higher concentration of nucleosomal DNA compared to hexanucleosomes as indicated by the absorption at 260 nm (inset). These data can be used to calculate

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the incorporation probability taking into account that the oligonucleosomes are inhomogeneous in brightness, since each complex may carry several EYFP tags. Assuming a stochastic incorporation of H2B-EYFP, the number of tags per complex obeys a binomial distribution and the amplitudes are changed in a defined way.24 Agarose gels (as in Figure 1(a)), absorption spectra, and FCS (as in Figure 3(a)) provide enough information to extract the incorporation probability for each sample of oligonucleosomes. Almost independent of fragment size, the incorporation probability is constant, with only the mononucleosomes showing systematic deviations (Figure 3(c)). Although these latter can be prepared with high yield and a homogeneous DNA length distribution, the content of fluorescent particles of fresh material gave unexpectedly high yet unstable values. This could be due to a considerable amount of unbound fluorescent histones, which might cosediment with this fraction in the sucrose gradient. Some variation for larger fragments ðN . 5Þ may reflect increasing uncertainty in determination of the nucleosome composition. Therefore, we averaged all values for fragments larger than mononucleosomes and obtained an incorporation probability of p ¼ 5ð^1Þ%: This number is in good agreement with that observed on gels and indicates that fluorescence is almost completely preserved during the isolation procedure. Brightness and mobility of H2B-EYFP in the cytoplasm The concentration and molecular brightness (intensity per particle) of freely diffusing H2BEYFP is determined via FCS in the cytoplasm of individual cells. The molecular brightness allows to transform intensities measured at a given laser power with CP or confocal imaging into absolute fluorophore concentrations. In the cytoplasm, a fluorescence intensity of up to 25 kHz (corresponding to a S/N-ratio of , 25) could be recorded with less than 10% loss due to photobleaching within a run of ten seconds. The average diffusion constant for H2B-EYFP in the cytoplasm is 7.3(^ 0.3) mm2/s ðn ¼ 62Þ: Figure 4 illustrates three modes of mobility: whereas at position 1 the shape of the correlation curve indicates free three-dimensional Brownian motion, at position 2 a typical long-tail correlation appears. Long-tail correlations are a result of locally confined movement.25,26 The observation of a more granular appearance of the cytoplasm compared to position 1 suggests that here membrane barriers, e.g. the Golgi apparatus or the endoplasmatic reticulum, are sensed. Nevertheless, significant initial bleaching occurred at some locations, preferably in the proximity of the nucleus. At position 3 the intensity is significantly increased and at the same time bleaching occurs at an initial phase of the intensity time trace, both of which is indicating binding of the fluorescent his-

Figure 4. H2B-EYFP in the cytoplasm. (a) Confocal images scanned with supplementary transmission of white light 3 mm above the cover slip at 0.2 mm pixel dimension (left panel) and in fluorescence mode with 0.1 mm pixel (right panel) performing FCS and CP at indicated positions (1, 2, and 3). (b) Autocorrelation curves as measured by FCS (six runs of ten seconds) with fit functions based on three-dimensional Brownian motion (red). Additional long-tail correlations (arrow) indicate obstructed diffusion at position 2. (c) Intensity time trace of a FCS-measurement (vertical broken lines divide into putative runs of ten seconds). The deviation in an initial phase from the exponential fit (red) indicates bleaching of immobilised fluorescence in the illumination volume. A typical background of non-fluorescent Hela cells is given as a reference (straight line).

tones to cellular structures (for detailed discussion of bleach curves see the following section). The average concentration of H2B-EYFP derived from fluctuation amplitudes of one to three measured positions in the cytoplasm of interphase cells is 146(^ 40) nM ðn ¼ 31Þ: During mitosis the concentration is significantly increased about threefold, presumably due to the fusion of the histone pools of cyto- and nucleoplasm (see below). Variations in molecular brightness of the fluorescent histones between cells were smaller than the variations of the concentration values themselves. For example, in a session of nine cells measured at constant laser power, the cytoplasmic

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concentrations varied with a standard deviation of 22%, whereas the molecular brightness varied only by 8% (data not shown). However, since it is not accurately known how global parameters (e.g. pH of the medium) or the local intracellular environment affect the fluorescence yield in a particular measurement, we used the averaged molecular brightness for each cell individually. A small fraction of histones diffuses relatively fast in the nucleoplasm The nuclear localisation sequences (NLS) of the histone domain target the fusion protein H2BEYFP into the nucleus, for details see Baake et al.27 and references therein. Within the nucleus, histones exist in at least two clearly distinguishable states of mobility: either they become incorporated via intact nucleosomes into the chromatin fibre, or

Figure 5. H2B-EYFP in the nucleus. (a) Three consecutive fluorescence images scanned with 0.1 mm pixel dimension 5 mm above the cover slip. Point wise fluorescence photobleaching (crosses) in the nucleolus (1) and a chromatic region (2) results in bleached spots (arrows). (b) Intensity time traces (black) from CP measurements at positions 1 and 2. The data are fitted to exponential functions (red) for bleach times .60 seconds. (c) Typical correlation functions as measured after bleaching show long-tail correlations (arrows) for obstructed diffusion at both locations.

Counting Nucleosomes in Living Cells

they belong to a pool of mobile histones in the nucleoplasm. We determined its concentration with CP:17 the laser focus was placed at a selected position and the fluorescence intensity recorded continuously over time. Bleaching of H2B-EYFP in a chromatic region results in a confined bleached spot (Figure 5(a)) as expected for a core histone with slow exchange.9,10 The CP signal first drops rapidly due to photobleaching of immobilised fluorophores in the illuminated spot and then turns into an exponential decay resulting from photobleaching of mobile fluorophores (Figure 5(b)). From the intercept of the fit curves and an experimentally defined correction factor (see Materials and Methods) the initial concentrations of mobile EYFP-molecules in the nucleoplasm were estimated to be on average 1.2(^ 0.4) mM ðn ¼ 29Þ: This is an eightfold increase compared to the cytoplasm. Interestingly, in nucleoli the free histone concentrations were found to be still about 80% with respect to chromatic regions of the same cells (compare left and right part of Figure 5(b)). The slight decrease could be a consequence of excluded volume effects inside the dense structures. Consecutive bleaching at different spots in the same nucleus steadily depleted the pool of mobile fluorescent histones, which indicates fast exchange between different areas (not shown). The mobility can be directly measured with FCS: correlation measurements in a series of bleached spots exhibit typical autocorrelation decay as expected for anomalous diffusion (Figure 5(c)). Diffusion induced correlation times were found in the range of a few milliseconds and thus prove the high mobility of a fraction of H2B-EYFP in the nucleoplasm. The effect of diffusion obstruction is apparently enhanced in the nucleolus, where the contribution of the “tail” is significantly increased (Figure 5(c), left), whereas in chromatin, the obstruction appears to be somewhat stronger than in the cytoplasm (compare Figure 5(b) right graph with Figure 4(b) at position 2). However, due to background-related uncertainties in FCS, we chose bleaching as a more reliable method to determine the concentration of mobile fraction of nuclear H2B-EYFP. The diffusion behavior of fluorescently tagged proteins in the nucleus and in the cytoplasm has been extensively studied in two of our recent publications.17,26 There, we found a pronounced diffusion anomaly for both free EGFP and H2BEYFP in the nucleus, but much less so in the cytoplasm. The average nucleosome density in a nucleus is , 140 mM The calculation of nucleosome concentrations is done in four steps. 1. Each pixel of the confocal section (Figure 5(a)) contains a photon count rate in kHz from the

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number of photon counts recorded during the residence time of the laser at the pixel. Dividing this number by the molecular brightness in kHz/particle at the same laser power yields the absolute numbers of particles generating the imaged signal. 2. Calibration of the observation volume with FCS (see Materials and Methods) allows transforming particle numbers into the absolute concentration of H2B-EYFP at each pixel. 3. CP in the nucleus yields the homogeneous background of rapidly diffusing fluorescent histones in the nucleoplasm. This level was globally subtracted from the image, obtaining the absolute concentration of immobilized H2B-EYFP at each pixel. 4. The measured concentration of fluorescent histones incorporated into chromatin was then converted into a nucleosome concentration: since p ¼ 5% of the H2B monomers are tagged with EYFP, 2p ¼ 10% of all nucleosomes carry at least one fluorescent tag. The nucleosome concentration cnuc is obtained from the fluorescent histone concentration cH2B-EYFP by cnuc ¼ cH2B-EYFP =2p: Confocal images of 28 nuclei have been calibrated, which lead to the distribution of mean values graphed in Figure 6(a). The average nucleosome density derived from nuclear cross-sections is 142(^ 28) mM for this population. For the interpretation of these data one has to bear in mind that the incorporation probability has been determined as an average for the whole population of cells (on isolated chromatin) and was applied for the calibration of nuclear cross-sections of individual cells. Thus, cellular fluctuations of the expression level of H2B-EYFP within the clone may broaden the width of the real distribution of average nucleosome concentrations in Figure 6(a). A direct measure for the expression level is the cytoplasmic concentrations of H2B-EYFP, which have been determined with FCS. Indeed, looking at individual cells, the average cytoplasmic concentrations correlate with the average nucleosome density derived from the nuclear cross-section of the same cell (a t-test on the correlation coefficient ðR ¼ 0:4779Þ gave a significance of p . 0:995 for positive correlation). Thus, the variation in average nucleosome concentration should in reality be even narrower than Figure 6(a) suggests. Figure 6(b) illustrates a nucleosome density map of a nucleus with a mean of 137 mM after calibration as an example not too far from the mean of the population. The concentrations fluctuate between 10 mM and 250 mM over distances of a few mm. The chromatin distribution shows typical spatial patterns: some parts of the dense chromatin are located in a fringe around the nucleolus or associate with the nuclear lamina. In nucleoli, concentrations of immobilised H2B-EYFP are strikingly low and lead to nucleosome concen-

Figure 6. Nucleosome density maps. (a) Histogram of the average nucleosome densities measured in calibrated confocal images. (b) Nucleosome density map of a typical nuclear cross-section. The grey values map concentrations from 0 mM to 250 mM. An exemplary profile illustrates variations along a straight line (bold).

trations of a few mM or even drop to zero in some nuclei (not shown). However, a global background correction based on CP in chromatic regions may cause some uncertainty for nucleoli, where the mobile pool of H2B-EYFP is diminished. Apart from the nucleoli, no nucleosome-free space could be detected in any of the imaged nuclei. Nucleosomes are smoothly distributed over the concentrations occurring in a nuclear cross-section In order to investigate whether nucleosomes can be grouped into distinct density classes, histograms of nucleosome concentrations were calculated for each of the calibrated images ðn ¼ 28Þ; and from there, the mass or numbers of nucleosomes distributed over the measured concentrations as found in the nucleosome density map (Figure 7). The nucleosome mass distribution is in all cases smooth with a single peak in the vicinity

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Figure 7. Normalised mass histogram for two exemplary nuclei (a) and (b). The data (circles) indicate the relative mass or numbers of nucleosomes found at a certain concentration within the nuclear cross-section (insets). A Gaussian function was fitted to the data (continuous lines). Deviations at low and high concentrations are indicated (arrows).

of the mean value. The central part of the histogram, representing the bulk of nucleosomes, has a Gaussian shape as confirmed by the fit curves in Figure 7. However, at the tails of the distribution the data show deviations. A shoulder appears at low concentrations for all of the imaged nuclei, which is most pronounced in nuclei with large nucleoli and therefore apparently large areas of low nucleosome concentrations (Figures 6(b) and 7(a)). A finite microscopic resolution at the nuclear border may also contribute to this effect. Deviations for a small sub-fraction of nucleosomes exist also at high concentrations: 25% of the imaged nuclei exhibit a small shoulder, which corresponds to a high content of chromatin dense regions (Figure 7(b)). However, we claim that there is only a weak statistical basis for the identification of chromatin dense (heterochromatic) regions in confocal images on the basis of absolute nucleosome densities.

Discussion Incorporation of fluorescent histones into the chromatin fibre The fluorescent histones of the transgenic Hela cells become a stable constituent of chromatin by integration into the nucleosome structure. They comprise 5% of the total amount of assembled

Counting Nucleosomes in Living Cells

H2B as determined in vitro. This number is an average over varying expression levels of the construct (less than ^ 20%), as well as all functional states during the cell cycle and every mechanism involved in nucleosome assembly. Assuming a binomial distribution of fluorescent tags along the fibre, one can conclude that on average every , 10th nucleosome carries at least one EYFP domain. In the solenoid model of the 30 nm chromatin fibre, 10 –12 nucleosomes form two turns. Furthermore, the nucleosomal repeat length in the cells of the transfected clone Y is 204 bp as compared to 185 bp in the wild-type cell. Thus, the linear mass density of core histones on the DNA molecule is reduced from 0.88 to 0.82 (by 2 6.5%), which overcompensates the deposition of supplementary mass of EYFP domains at the histone cores (þ 2.6% assuming constant linker length). This suggests that steric reasons may play a role for the increased spacing. It is interesting to note that the additional volume and charge of the autofluorescent protein affects the geometry of the chromatin fibre as a whole, not only the positioning of tagged nucleosomes: asymmetric linker lengths only for a fraction of nucleosomes would broaden the DNA fragment length distribution obtained by MNase treatment rather than shifting its mean values. It seems that nucleosome spacing in vivo is maintained on length scales larger than the distance of two adjacent nucleosomes. However, according to current models for chromatin remodelling mediated by multi-enzyme complexes of the ISWI subfamily, e.g. NURF, CHRAC, or SWI/SNF, remodelling seems to act at individual nucleosomes by facilitating sliding along the DNA.28 If so, our data suggest that the higher order structure of a putative 30 nm fibre favours energetically the regular repeat length instead of tolerating local disorder as introduced by EYFP domains. H2B-EYFP in the cytoplasm The central step in our approach is to link fluorescence intensities to particle numbers. With FCS, the fluorescence intensity and particle concentration of a solution of fluorophores are measured independently, making the method suitable to calibrate imaged signals. The molecular brightness was determined in the cytoplasm where nascent fluorescent histones are accessible. The decay of the correlation curves is related to the mobility, whereas the amplitude yields the concentration and molecular brightness of the diffusing proteins. The variance of average concentrations derived from fluctuation amplitudes, signifies with 27% an upper estimate for the variation in expression level of the fusion protein. In contrast, the molecular brightness varied less and could be determined with an error of 10% for individual cells. We assume that the observed particles represent single H2B-EYFP molecules, possibly in complexes with non-fluorescent endogenous components for

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the following reasons: (1) mitotic cells containing threefold concentration of mobile fluorescent histones showed the same molecular brightness as interphase cells; (2) if we assume according to the incorporation probability that 5% of the cytoplasmic H2B are fluorescent, a fraction of putative H2B-EYFP dimers would still remain below 5% of the total fluorescence independent of their association constant, which is negligible; (3) there is good experimental evidence that histones in the cytoplasm diffuse in complexes with a multitude of other endogenous, and hence, non-fluorescent molecules. Recently, it has been reported that transport of the core histones is receptor mediated and energy dependent.27 The authors report that the central globular domain alone, lacking a classical NLS, is sufficient to guide a genetically fused reporter protein into the nucleus, thus, histones may be also transported as heteronomous multimers. Furthermore, a number of histone-binding proteins have been isolated from chromatin assembly competent cellular extracts. For example, NAP-1 preferentially binds H2A and H2B and has been proposed to function as a histone shuttle.29 H2B-EYFP in the nucleus Bleaching in combination with FCS in bleached spots showed that a small fraction (, 1%) of nuclear H2B-EYFP obey a similar mobility as nascent H2B-EYFP molecules in the cytoplasm. The nucleoplasmic pool is about eightfold as concentrated compared to the cytoplasm and characterises quantitatively the efficiency of the nuclear transport of H2B. The nucleoplasmic fluorescent histones move freely around the chromatin fibre, which shows large concentration fluctuations within length scales of some mm. For several reasons, we assign these spatial fluctuations to be a physical property of the polymeric nature of chromatin rather than biological diversity at particular loci of the DNA. (1) The statistical incorporation of the fluorescent histones could be verified in vitro for dinucleosomes up to octamers. Assuming that the mechanism of nucleosomes assembly is a locally confined process involving not more than a few neighbouring nucleosomes on the DNA, it is very unlikely, that the distribution of fluorescence along the fibre is clustered because of differences in incorporation probability. (2) The nucleosome density maps reflect typical patterns described for the chromatin distribution in interphase, e.g. the association of dense regions with the nucleoli or the nuclear lamina. (3) H1-EYFPexpressing cells show a similar fluorescence pattern in the nucleus (data not shown), indicating that the distribution of H2B-EYFP correlates with the distribution of binding sites for H1 and hence with the distribution of real nucleosomes. (4) A concentration of 100 mM at a certain spot still implies the presence of , 104 nucleosomes in the observation volume. Local deviations in fluorescent histone incorporation, e.g. on the level of a

gene with several hundreds of nucleosomes, should not severely change the results. (5) The nucleosome density maps of the population yielded mean nucleosome concentrations of 140 mM. To cross-check this result, we took 3D-stacks of the nuclei and estimated the nuclear volume. Although there is some uncertainty due to the threshold setting we obtained volumes of about 1000 mm3. Assuming a repeat length of 200 bp and a tetraploid genome (4n) of 7 £ 109 bp DNA as determined from DNA extraction, the average nucleosome concentration would be , 116 mM which is in good agreement with the fluorescence determined concentrations. The nucleosome density may be converted into the density of a chromatin fiber with the canonical packing of six nucleosomes per 11 nm and a fibre diameter of 30 nm. From this follows a volume per nucleosome of 1296 nm3, corresponding to a molar concentration of 1.28 mM at a packing density of 100%. In a hexagonal dense packing of cylinders, the volume occupation is 90.6%, thus the maximum attainable nucleosome concentration would be 1.16 mM. The nucleosome densities measured here correspond therefore to chromatin fiber volume fractions of about 10%. This is averaged over an observation volume of 0.3 mm £ 0.3 mm £ 1.2 mm ¼ 0.056 mm3, or 104 nucleosomes, thus locally nucleosome packing densities could still vary substantially. These estimates of chromatin concentrations allow to draw conclusions about the “mesh spacing” of the chromatin fibre. Again taking the hexagonal dense packing at the maximum concentration of 1.16 mM, the average distance l between the fibre centres would be 30 nm, and would vary inversely proportional to the square root of the pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi concentration c as: l ¼ 30 nm= c½M=1:16 mM: Accordingly, a concentration of 100 mM corresponds to a mesh spacing of 102 nm. The most dense areas with concentrations of up to 260 mM feature a mesh spacing of 63 nm, still significantly larger than the typical size of diffusing protein complexes. Heterochromatin is regarded as a condensed and repressed state. However, looking at the large mesh spacing (, 100 nm) estimated even for the highest concentrations (, 260 mM) found, it seems that all nuclear locations are accessible for diffusing protein complexes, in agreement with recent in vivo mobility studies.7,26 Therefore, a regulation mechanism for the accessibility of chromatin binding sites by condensation probably takes place on smaller length scales than can be resolved with light microscopy.

Materials and Methods Construct and cell line The human histone H2B gene (Acc# X57127) was amplified by genomic PCR and inserted N-terminal of

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enhanced cyan fluorescent protein (ECFP) into the promoter less plasmid pECFP-1 (Clontech). Upstream we inserted the HindIIIc fragment of simian virus 40 (SV40) in reverse direction, such that the fusion protein of 372 amino acid residues is expressed through the early SV40 promoter. In a second step, ECFP has been replaced by its spectral relative EYFP (Clontech). The linker between H2B and EYFP contains seven amino acid residues (RDPPVAT) originating from remaining bases of the multiple-cloning site. Transfection was carried out with Lipofectamin (Life Technologies) as proposed by the manufacturer. Cells were grown in a 5% CO2 humidified atmosphere and passaged in RPMI 1640 without phenol red (Life Technologies) supplemented with 10% (v/v) foetal calf serum. A monoclonal cell line (Y) was selected by G418 (Life Technologies). For in vivo imaging, cells were cultivated sub-confluent on chambered cover slips (Nunc) and mounted on the scanning stage at room temperature. Cell cycle analysis Prior to fluorescence-activated cell sorting (FACS), the cells were fixed in 100% ice-cold ethanol and exposed to 5 mM DAPI (4,6-diamidino-2-phenylindole-2HCl, Serva) and 5 mM SR101 (Sulforhodamine 101; Eastman Kodak). The measurements were done in a Cytofluorograph 30L (Ortho Diagnostics Systems Inc.) using the UV lines (351 – 364 nm) of an Argon laser. DAPI emission was collected above 450 nm and the data processed according to Stoehr et al.30 Isolation of oligonucleosomes Intact nuclei were isolated by means of mild lysis of the plasma membrane: Petri dishes (Ø ¼ 15 cm) with confluent grown cells were washed with 10 ml isotonic Tris (25 mM Tris-HCl, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5 mM MgCl2, 0.3 mM Na2HPO4, pH 7.4) and twice with 5 ml isotonic Hepes buffer (50 mM Hepes, 220 mM sucrose, 1 mM EDTA, 1 mM DTT, pH 8). The cells were scraped and collected in a tube. The volume was adjusted to 10 ml with isotonic Hepes and a final concentration of 0.25% Nonidet P-40. Lysis was supported by mechanical agitation on a vortex mixer. The broken cells were diluted to 30 ml with isotonic Hepes, centrifuged at 1000 rpm at 4 8C, and carefully resuspended in reticulocyte standard buffer RSB (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH 7.4). The nuclei were stored in 50% (v/v) glycerol at 270 8C for several months. The DNA concentration was determined according to Lawson et al.31 Prior to chromatin preparation, an aliquot of nuclei containing 2 mg of DNA was dialysed against 500 ml RSB for two hours at 4 8C, washed in 50 ml RSB, and centrifuged for five minutes at 1500 rpm, 4 8C. The soft pellet was dissolved in 10 ml RSB and digested with MNase (Amersham; 1.5 U/ml) in the presence of 1 mM CaCl2 for ten minutes at 37 8C. After stopping the reaction with 5 mM EGTA, nuclei were pelleted again and dissolved in 250 ml of 0.2 mM EDTA for hypotonic swelling ten minutes on ice. Lysis was completed with 1% Nonidet P-40 by means of rigorous agitation for 90 seconds on a vortex mixer. Less than 200 ml were loaded onto 13 ml of 10% to 30% (w/v) sucrose gradients containing 100 mM NaCl and sedimented in a preparative ultracentrifuge for 15 hours at 40,000 rpm, and 4 8C (Beckmann L-8M, SW-41). Fractions (500 ml) of the gra-

Counting Nucleosomes in Living Cells

dient were collected and pooled with respect to DNA length. To remove sugar, the native oligonucleosomes were dialysed against TE20 (10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 20 mM KCl) and concentrated with spin columns (PSE membrane, Viva Science). The samples were stored at 4– 8 8C and measured within a few weeks. Gel electrophoresis and immunoblotting Each fraction (10 ml) of the sucrose gradient was digested with 10 mg/ml Proteinase K (Sigma) and analysed with electrophoresis on 1% (w/v) agarose gels in TBE. Band sizes and intensities were quantified with an image-evaluation-software (Intelligent Quantifier, Bio Image Systems Corporation). The average number of bound nucleosomes per complex was calculated accorP ding to Napp ¼ i ri Ni : This was only used to determine the incorporation probability with FCS (see below). ri designates the quantified fraction of a DNA band as present on the agarose gel weighing the assigned number of nucleosomes Ni of the particular fragment length (using the repeat length of 204 bp). Nuclear proteins and isolated nucleosomes were resolved with discontinuous SDS-PAGE on 0.75 mm thick mini-gels (BioRad), with a 5% stacking phase layered on top of a 18% separation gel. Proteins were stained with Coomassie32 and silver33. H2B-EYFP was immunodetected using a polyclonal rabbit anti-XFP horseradish peroxidase conjugate (Clontech, #8369-1). Fluorescence fluctuation microscopy (FFM) For FCS, CP, and confocal imaging we used a homebuilt setup, the fluorescence fluctuation microscope, FFM.17 FFM combines an FCS module23 and a beam scanning unit attached to the video port of an inverted microscope (IX-70, Olympus). The EYFP fluorescence was excited with the 488 nm line of an Ar –Kr laser (Omnichrome) and detected from 515 nm to 545 nm with avalanche photodiodes (SPCM-AQR-13, Perkin– Elmer) in photon counting mode. Appropriate dichroic mirrors and filters were used for spectral separation and selection. The control software written in our laboratory allows on the one hand to acquire confocal fluorescence images as well as white light transmission images and on the other hand to position the laser with 30 nm precision for FCS and CP. For FCS and CP, the detector signal is fed into an ALV-5000/E correlator card (ALV Laser GmbH, Langen, Germany), which records the signal versus time and calculates its autocorrelation function simultaneously. Intracellular measurements with the FFM To determine the concentration and the molecular quantum yield, white light transmission images were used to select up to three positions randomly in the cytoplasm at which we performed correlation measurements of one minute (six of ten seconds). Before investigating the fluorescent nuclei with CP and imaging, we adjusted the illumination intensity such that the fluorescence yield in the cytoplasm was between 0.3 kHz and 0.7 kHz per particle corresponding to a laser power between 0.5 kW/cm2 and 1 kW/cm2. Varying the laser power up to a factor of 2 did not affect the measured concentration values. Eventually, the first run was abandoned if distorted by an apparently immobilised fraction of intensity usually vanishing within a few

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Counting Nucleosomes in Living Cells

seconds due to photobleaching. The z-position was defined visually by focussing the laser light to the upper surface of the cover slide, recognised by a pointlike reflex. This process caused an uncertainty of ^1 mm in combination with the positioning accuracy of our stage. The focus was placed at z ¼ 4(^ 1) mm above the cover slide. Intensity z-scans proved that such a focal depth is usually positioned inside the cytoplasm. FCS analysis The measured autocorrelation curves GðtÞ were analysed as described.26 For fluorescein (Fluorescein 5/6C; Molecular Probes), we obtained two correlation times corresponding to the relaxation of the triplet state population of the fluorophore34 and to the mean dwell time of the dye molecule in the focus ðtdiff Þ; respectively. For intracellular H2B-EYFP we obtained the amplitude Gð0Þ as well as two correlation times due to the (de-) population of two non-fluorescent states in a protonation equilibrium20,21 and a diffusion induced correlation time. From the latter we calculated diffusion constants using the measured dwell time of fluorescein and its published diffusion constant,35 2.6 £ 10210 m2/s. In some cases of intracellular measurements, a second diffusional correlation time was taken into account, which is one appropriate way to cope with obstructed diffusion in dense meshworks or confined volumes.26,36 The observation volume has been determined by means of calibrating the amplitude of a fluorescein standard (the amplitude is much more sensitive to optical alignment than the diffusion time) with a solution of EYFP isolated from cells, which was characterised by absorption and FCS. The effective observation volume was confirmed by the diffusional correlation time tdiff of fluorescent polystyrole 60 nm beads (Bangs Laboratories) and applying Veff ¼ kð4pDst tdiff Þ3=2 ; where Dst denotes the diffusion coefficient of the beads (7.2 £ 10212 m2/s) and the factor k ¼ 4 characterises the elongated geometry of the focus. The observation volume was typically 0.3–0.7 fl. When evaluating the intracellular autocorrelation curves, the correlation times of non-fluorescent states were fixed to 30 ms and 300 ms, which are representative values for oligonucleosomes in vitro. In vivo-values for the dark states could not be determined due to noise at short correlation times. However, their manual variation in the fitting process did not affect the intercept of the correlation significantly. For all correlation measurements, a background of autofluorescent components was taken into account as determined in non-transfected HeLa cells. There, the background intensity varied from 0.6 kHz to 2 kHz depending on laser power. FCS measurements of fluorescent oligonucleosomes in vitro: how statistical association of fluorophores at a substrate affects correlation functions has been described recently.24 The amplitude is changed to Gð0Þ ¼ ð1 2 1=n þ 1=npÞ=cVeff ; where n is the number of binding sites, p the probability to find a bound fluorophore and c the total concentration of labelled substrate. In the case of oligonucleosomes, the number of possible incorporation sites is twice the number of nucleosomes, n ¼ 2Napp ; while the incorporation probability p is given by the fraction of fluorescent histones with respect to the total content of H2B. The concentration c was measured independently using the absorption at 260 nm and the molecular weight MW ¼ repeat lengthðbpÞNapp 660 Da/bp. Inserting this into the upper equation, p can be written as a function of determined quantities: p ¼ 1=ð2Napp cVeff Gð0Þ2 2Napp þ 1Þ:

Image analysis Image properties are determined by three parameters: the size of the scanned area, the pixel distance (PD), and the residence time of the laser at each pixel (PT). Images in the transmitted light mode were taken with PD ¼ 0:2 mm, while fluorescence images were scanned at PD ¼ 0:1 mm resulting in two- to threefold over sampling. For fluorescence images, we selected PTs between 100 ms and 230 ms in order to use the available range of 255 counts per pixel without saturation effects. From FCS measurements in the cytoplasm the concentration c of the fluorophores and therefore the intensity of a 1 M solution could be calculated according to F~ ¼ F=c (kHz/M), where F is the mean intensity obtained in the measurement at a certain laser power. The background of mobile fluorescence in the nuclei was determined with CP for 120 seconds in chromatic regions. CP in fixed cells showed that half of the intercept Fdiff;0 of an exponential fit to the last 60 seconds of the recording reflects the concentration of mobile fluor~ The counts in the escence according to cdiff ¼ 0:5Fdiff;0 =F: images were transformed into concentrations of immobi~ 2 cdiff : A factor lised EYFP cimmo ¼ countsxy;image =ðPT FÞ of 1=2p transforms into concentrations of nucleosomes. For further analyses, such as line profiles and histograms, NIH ImageJ V1.30h was used.

Acknowledgements We thank Michael Sto¨hr for performing the FACS analysis, Gaby Bergmann and Nathalie Brun for their help in Western blotting and Malte Bussiek for critical reading of the manuscript. This work was supported by the Volkswagen foundation as part of the program “Physics, Chemistry, and Biology with Single Molecules”.

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Edited by W. Baumeister (Received 27 May 2003; received in revised form 19 August 2003; accepted 19 August 2003)

Supplementary Material for this paper is available on Science Direct.