Biochemical analysis of chromatin structure and function using Drosophila embryo extracts.

The biochemical analysis of chromatin structure and function is greatly facilitated by the availability of cell-free systems that assemble chromatin under physiological conditions. One such system that has shown great potential is derived from extracts of early Drosophila embryos. These embryos contain large maternal stocks of chromatin constituents, such as histones and assembly factors. Chromatin assembled in these extracts resembles native chromatin in many respects: it displays physiological nucleosome repeat lengths, it is complex, containing a wealth of nonhistone proteins as well as enzymatic activities, and it has dynamic properties that allow the interaction of DNA-binding proteins that regulate important cellular processes. Most importantly, chromatin with variant properties, e.g., with respect to the basic geometry of the nucleosomal array, histone modifications, and its content of linker histones or nonhistone proteins, can be obtained by manipulating the reconstitution conditions. The synthesis of uniform chromatin with specific characteristics should allow the analysis of the functional significance of the structural and biochemical heterogeneity observed in vivo.

RNA polymerase I transcription on nucleosomal templates: the transcription termination factor TTF-I induces chromatin remodeling and relieves transcriptional repression.

Eukaryotic ribosomal gene promoters are preceded by a terminator element which is recognized by the transcription termination factor TTF-I. We have studied the function of this promoter-proximal terminator and show that binding of TTF-I is the key event which leads to ATP-dependent nucleosome remodeling and transcriptional activation of mouse rDNA pre-assembled into chromatin. We have analyzed TTF-I mutants for their ability to bind to free or nucleosomal DNA, and show that the DNA binding domain of TTF-I on its own is not sufficient for interaction with chromatin, indicating that specific protein features exist that endow a transcription factor with chromatin binding and remodeling properties. This first analysis of RNA polymerase I transcription in chromatin provides a clue for the function of the upstream terminator and establishes a dual role for TTF-I both as a termination factor and a chromatin-specific transcription activator.

The effect of nucleosome phasing sequences and DNA topology on nucleosome spacing.

The distances between the nucleosomes in eukaryotic chromatin that define the nucleosome repeat length are not universally constant, but vary between different cell types and activity states. We have previously established in a cell-free system that nucleosome spacing is essentially governed by electrostatic principles, most likely through charge neutralisation of linker DNA by cations either free in solution or on flexible histone domains. On the basis of the tight correlation between the parameters that affect nucleosome spacing and those that influence the folding of the nucleosomal fiber into higher order structures, we suggested that there is an intimate relationship between nucleosome spacing and chromatin folding. Here we describe DNA topology as a new parameter that influences nucleosome spacing in a predictable way. The effects of topology and cation concentrations integrate to define the final repeat length. The phenomenon of "nucleosome phasing" describes nucleosomal arrays that are generated through positioning of nucleosomes by the underlying DNA sequence. To determine the relative contribution of DNA sequence and the parameters intrinsic to physiological chromatin for nucleosomal positions, we created situations where these two principles were in conflict. We found that nucleosome repeats directed by a strong positioning sequence are dominated by the cation-induced spacing as well as by the effects of topology. We conclude that the DNA sequence effects nucleosome spacing only by "fine tuning" of nucleosome positions within the framework of a repeat pattern that is established by other principles.

Electrostatic mechanism of nucleosome spacing.

Native bulk chromatin is characterized by regular arrays of nucleosomes with defined internucleosomal distances. The nucleosome repeat length is not a constant but varies between species and cell-types, during differentiation and during gene activation. Previous studies have highlighted the importance of linker histones as a major determinant of nucleosome repeat length in vivo. We used a physiological reconstitution system derived from Drosophila embryos to study nucleosome spacing. In these extracts, histone H1 incorporation increases the apparent linker length in a gradual way. Manipulation of the chromatin assembly conditions in vitro allowed us to define additional parameters that modulate nucleosomal distances, such as protein phosphorylation events and the precise ionic conditions during the reconstitution. Interestingly, moderate changes in the concentrations of mono-, di-, and multivalent cations affect the precise distances between nucleosome cores remarkably. These changes in the ionic environment are unlikely to affect the association of linker proteins but are known to influence the folding of the nucleosomal fiber by modulation of electrostatic forces. Our results suggest electrostatic interactions in chromatin units as major determinants of nucleosome spacing. Nucleosome spacing and the folding of the nucleosomal fiber can therefore be explained by common principles, most notably the neutralization of charges in linker DNA.

Energy-dependent chromatin accessibility and nucleosome mobility in a cell-free system.

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile. Nucleosome movements, most likely their sliding, occurred even in the presence of the linker histone H1. The dynamic state of chromatin in the presence of the activity and ATP globally increased the accessibility of nucleosomal DNA to incoming proteins. This unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle which is likely to be fundamental to the mechanism of chromatin remodelling and the binding of regulatory proteins.