Transport of nucleosome core particles in semidilute DNA solutions

Abstract

We studied the diffusion of native and trypsinized nucleosome core particles (NCPs), in aqueous solution and in concentrated DNA solutions (0.25-100 mg/ml) using fluorescence correlation spectroscopy (FCS). The highest DNA concentrations studied mimic the DNA density inside the cell nucleus. The diffusion coefficient of freely diffusing NCPs depends on the presence or absence of histone tails and is affected by the salt concentration due to the relaxation effect of counterions. NCPs placed in a network of long DNA molecules (30-50 kbp) reveal anomalous diffusion. We demonstrate that NCPs diffusion is in agreement with known particle transport in entangled macromolecular solutions as long as the histone tails are folded onto the particles. In contrast, when these tails are unfolded, the reversible adsorption of NCPs onto the DNA network has to be taken into account. This is confirmed by the fact that removal of the tails leads to reduction of the interaction between NCPs and the DNA network. The findings suggest that histone tail bridging plays an important role in chromatin dynamics.

FIGURE 1

SDS-15% PAGE gel electrophoresis, stained with Silver. For (A) native nucleosome core particle and (B) after trypsin digestion: The bands are shifted to lower molecular weight

FIGURE 2

(A) Experimental autocorrelation functions, g₂(t) recorded for intact NCPs for two different salt concentrations: C_s = 10 mM (up triangle) and 200 mM (open square). The functions, normalized by the number of particles, N, inside the effective laser volume are fitted according to Eqs. 2 and 5 (continuous lines). Inset zoom of the g₂(t) function for time ranging from 200 to 600 ms (B) Residual between the fit and experimental functions for C_s = 10 mM.

FIGURE 3

(A) Variation of the diffusion coefficient and (B) the hydrodynamic radius as a function of the salt concentration, C_s, for intact and trypsinized NCPs. Both kind of particles are labeled with Toto-3, with in average one particle over ten labeled with one dye. Experiments were performed at a NCP concentration C_NCP = 0.1 mg/ml.

FIGURE 4

Autocorrelation function recorded for C_s = 10 mM, in a DNA concentrated solution of 5 mg/ml. The particles were labeled with Toto3, with in average one over ten particles labeled with one dye. The experiments were performed at very low NCP concentration: C_NCP = 0.05 mg/ml. The curve is fitted according to the Eqs. 5 and 6 when the anomalous parameter β is fixed to 1 (dashed line) and optimized to 0.76 (continuous line).

FIGURE 5

(A) Variation of the diffusion coefficient of intact NCPs as a function of the DNA concentration, normalized by D₀, the diffusion coefficient of free NCPs. The data are recorded for three salt concentrations: C_s = 10, 100, and 400 mM. (B) Variation of the anomalous parameter β resulting from the fit of the autocorrelation function by using Eqs. 5 and 6. The lines are just guides for the eyes.

FIGURE 6

Diffusion coefficient recorded for trypsinized NCPs, as a function of the DNA concentration and for two different salt concentrations: C_s = 10 and 100 mM. By comparison, the diffusion coefficient measured for intact NCPS for C_s = 10 mM, is also plotted.