ABSTRACT Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca^sup 2+^ and Mg^sup 2+^) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg^sup 2+^) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.
INTRODUCTION
Nucleosome core particles are the structural units of eukaryotic chromatin. They are formed by the association of a 146-bp DNA fragment coiled around a protein octamer composed of four different histones (H2a, H2b, H3, H4). The particle has the shape of a cylinder, 110 A in diameter and 60 A thick. Its structure has been determined with high resolution (LUger et al., 1997; Harp et al., 2000) with the exception of parts of the histone tails, which are highly positively charged and protrude from the particle. These nucleosome core particles are linked together by DNA to form ordered nucleosomal arrays, which are themselves highly compacted into chromatin by association with HI histones and other proteins. However, chromatin is not a homogeneous and frozen structure. Cells regulate chromatin folding both temporally and spatially, and histones are dynamic components involved in this regulation through posttranslational modifications (including acetylation, phosphorylation, methylations, etc.) which may take place on the histone tails. Many proteins have been shown to be involved in this remodeling of chromatin, which is suspected to be of great importance in the regulation of transcription or induction of mitosis for instance (Strahl and Allis, 2000). The compaction of chromatin arrays has also been extensively studied in vitro. It has been demonstrated that the polyelectrolyte character of DNA, nucleosome, and chromatin was responsible for the compaction of the fiber (Widom, 1986; Clark and Kimura, 1990). It was shown also that the condensation of the fiber can be achieved by addition of cations in the absence of HI histones, but the integrity of the histone tails is absolutely required (Fletcher and Hansen, 1996; Widom, 1998). The presence of divalent cations is also necessary to reach the ultimate states of condensation of the fiber. Nevertheless, numerous questions remain open due to the complexity of the system.
In the present work we investigate the polyelectrolyte properties of solutions of isolated nucleosome core particles over the range of ionic conditions maintaining the stability of the nucleoprotein complexes. NCP can be viewed as colloids whose charges are heterogeneously distributed at the surface of the particle: negative charges carried by the DNA phosphate groups and positive charges carried by the lysine and arginine residues on the histone tails. We analyzed the effects of divalent cations Mg^sup 2+^ and Ca^sup 2+^, which are widely distributed in biological systems and play an important role in many enzymatic activities related to replication, transcription, and recombination. These divalent cations can induce the compaction of the chromatin fiber (Widom, 1986) but are inefficient in condensing pure DNA in aqueous solution (Bloomfield et al., 1994, 2000). We show that both cations may induce the aggregation of isolated NCP under defined ionic conditions and we question the reasons for this aggregation. Indeed, the stability of the solutions of negatively charged polyelectrolytes in the presence of added multivalent salts depends on the chemical nature of the functional groups carrying the polyion charges and on their interaction with the cations. Two different mechanisms have been proposed to explain the aggregation phenomenon observed at low ionic strength in solutions of polyelectrolytes (Oosawa, 1971; Record et al., 1978). They correspond to two extreme cases depending on the value of the chemical affinity constant between the charged groups and the cations. For a low affinity, the electrostatic interaction leads to the counterion condensation in the vicinity of the macroion. In this case, the aggregation phenomenon is due to the electrostatic interaction resulting from the counterion condensation. On the opposite, for a strong affinity, a specific interaction of the multivalent cation at a particular binding site of the polyelectrolyte leads to the formation of a complex. This chemical association is thought to produce a hydrophobic complex by dehydration of the cation and of the charged group (Sabbagh and Delsanti, 2000). For an intermediate value of the affinity constant, in a static approximation, site-specific binding and condensed states can coexist.
The conditions of precipitation of nucleosome core particles have been determined experimentally and compared to the previous results obtained with spermine (4+) (Raspaud et al., 1999). The role of the electrostatic interactions in the aggregation phenomena is analyzed. Moreover, we have investigated by electrophoretic measurements the effects of the addition of multivalent cations on the effective charge of the aggregates, which let us get information on the repulsion between the nucleosome core particles, for the different added salt concentrations.
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[Author Affiliation]
Marta de Frutos, Eric Raspaud, Amelie Leforestier, and Fran(;oise Livolant Laboratoire de Physique des Solides, Universite de Paris Sud, 91405 Orsay Cedex, France
[Author Affiliation]
Received for publication 27 December 2000 and in final form 20 April 2001.
[Author Affiliation]
Address reprint requests to Dr. Marta de Frutos, Laboratoire de Physique des Solides, Universit6 de Paris Sud, bat 510, 91405 Orsay Cedex, France. Tel.: 33-1-6915-5380; Fax: 33-1-6915-8004; E-mail: defrutos@lps.u-psud.fr.
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