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The PHD finger and the bromodomain are small protein domains that occur in many proteins associated with phenomena related to chromatin. The bromodomain has been shown to bind acetylated lysine residues on histone tails. Lysine acetylation is one of several histone modifications that have been proposed to form the basis for a mechanism for recording epigenetically stable marks in chromatin, known as the histone code. The bromodomain is therefore thought to read a part of the histone code. Since PHD fingers often occur in proteins next to bromodomains, we have tested the hypothesis that the PHD finger can also interact with nucleosomes. Using two different in vitro assays, we found that the bromodomain/PHD finger region of the transcriptional cofactor p300 can bind to nucleosomes that have a high degree of histone acetylation. In a nucleosome retention assay, both domains were required for binding. Replacement of the p300 PHD finger with other PHD fingers resulted in loss of nucleosome binding. In an electrophoretic mobility shift assay, each domain alone showed, however, nucleosome-binding activity. The binding of the isolated PHD finger to nucleosomes was independent of the histone acetylation levels. Our data are consistent with a model where the two domains cooperate in nucleosome binding. In this model, both the bromodomain and the PHD finger contact the nucleosome while simultaneously interacting with each other.

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro., (Copyright 1999 Academic Press.)

The hepatocyte nuclear factor-1 (HNF1) is a homeodomain transcription factor that binds DNA as a dimer. HNF1 dimers associate with two molecules of DCoH, a bifunctional protein that also has an enzymatic function in the tetrahydrobiopterin regeneration, to form stable heterotetramers also capable of DNA binding. Employing purified, recombinant HNF1, HNF1/DCoH heterotetramers and DCoH homotetramers we investigated whether DCoH affects interactions of HNF1 with nucleic acids. Although we detected no direct binding of DCoH to DNA or RNA, DCoH stabilized HNF1/DNA complexes and promoted interactions with sub-optimal DNA target sequences such as the human alpha1-antitrypsin TATA box region. Importantly, we also observed interactions of HNF1 with RNA, but these interactions were completely abolished when HNF1 was complexed with DCoH. Interestingly, DCoH retains its enzymatic activity while complexed with HNF1. Our results document intermolecular regulation of HNF1 binding to nucleic acids by DCoH.

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.

Transcriptional activation of the Drosophila hsp27 gene in response to heat shock critically relies on binding sites for heat shock factor (HSF) about 300 bp upstream of the transcription start site. In contrast to the well-characterised heat-inducible hsp70 and hsp26 promoters, no other transcription factor binding sites have been identified closer to the TATA box. In order to understand the structural requirements for activation from a distance we studied the protein-DNA interactions at the hsp27 promoter in Drosophila embryos and tissue culture cells before and after heat induction. Genomic footprinting with nucleases and a chemical probe, the 1,10-phenanthroline cuprous complex (OP-Cu), suggests that the DNA between the TATA box and the heat shock elements (HSEs) is constitutively organised by a positioned nucleosome, effectively shortening the distance between the distal HSEs and the TATA box. Protection of the TATA element from nuclease attack and the OP-Cu reactivity pattern around the start site of transcription is consistent with the constitutive presence of TFIID and the "poised polymerase", a transcription machinery blocked in an early phase of elongation. The general transcription factors at the TATA box and the positioned nucleosome are separated by a stable structure, presumably a protein bound to a palindromic sequence. These constitutive features define the "preset" architecture of the promoter within which the induced binding of HSF in vivo is observed. Our study highlights the importance of positioned nucleosomes as architectural elements within promoters and identifies a new regulatory sequence that may function either to direct a nucleosome boundary or to mediate signals of distant activator proteins.

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.