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P B Becker

Publications and source records attributed to P B Becker.

At least 19 recordsLinked to original sources
PubMed1997

The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids.

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.

Animals↗
PubMed1997

Genomic footprinting of Drosophila embryo nuclei by linker tag selection LM-PCR.

The unmatched power of Drosophila genetics revealed the complex regulatory network of gene activities that governs the development of higher eukaryotes. An understanding of gene control at the level of transcription requires insight into the protein/DNA interactions that regulate transcription in the developing embryo. Genomic footprinting allows the direct visualization of these protein/DNA interactions within intact nuclei or cells. In combination with other in vivo assays such as protein/DNA crosslinking and classical biochemistry, genomic footprinting can give valuable insight into the architecture of promoters in various states of activity. In this article we summarize our experience in analyzing Drosophila embryos by genomic footprinting and describe modifications of the ligation-mediated PCR procedure that have improved this analysis. Applications of genomic footprinting to embryos are currently limited by the fact that all target nuclei must be uniform with respect to the protein/DNA interactions at the chosen site. We discuss strategies that should allow the analysis of small numbers of cells derived from heterogeneous populations and tissues.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

The architecture of the heat-inducible Drosophila hsp27 promoter in nuclei.

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Transcription factor-mediated chromatin remodelling: mechanisms and models.

The association of DNA with nucleosomes in chromatin severely restricts the access of the regulatory factors that bring about transcription. In vivo active promoters are characterised by altered, almost transparent chromatin structures that allow the interaction of the transcriptional machinery. Recently, enzymatic activities have been discovered that facilitate the binding of transcription factors to chromatin by modifying nucleosomal structures in a process that requires energy. The mechanisms by which chromatin is remodelled may involve nucleosome movements, their transient unfolding, their partial or even complete disassembly. The dynamic properties of chromatin that underlie these structural changes are fundamental to the process of regulated gene expression.

Chromatin↗
PubMed1995

Dual regulation of the Drosophila hsp26 promoter in vitro.

Efficient heat shock induction of Drosophila hsp26 gene transcription in vivo requires binding sites for heat shock factor (HSF) and GAGA factor (GAF) close to the TATA box (proximal elements) as well as 350 bp upstream of the start site of transcription (distal elements). We have evaluated the contribution of hsp26 promoter sequences to transcriptional activity in extracts from either heat shocked or unstressed fly embryos. Efficient transcription in either extract was governed by distinct regulatory principles. Transcription in extracts from unstressed embryos relied solely on GAGA elements which efficiently counteracted repression by abundant non-specific DNA-binding proteins. Transcription in extracts from heat shocked embryos depended only a little on GAGA elements, relying mainly on functional HSEs. Constitutively active recombinant HSF or native factor in an extract from heat shocked embryos was able to truly activate transcription essentially via proximal HSEs, but not when bound to distal sites. These two modes of regulation in vitro may correspond to the two functional states of the promoter before and after heat shock in vivo.

Animals↗
PubMed1995

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.

Adenosine Triphosphate↗
PubMed1995

Chromatin remodeling by GAGA factor and heat shock factor at the hypersensitive Drosophila hsp26 promoter in vitro.

The chromatin structure at the Drosophila hsp26 promoter in vivo is characterized by two DNase I-hypersensitive (DH) sites harboring regulatory elements. Proximal and distal DH sites are separated by a positioned nucleosome. To study the contribution of transcription factors to the establishment of this specific chromatin configuration we assembled nucleosomes on the hsp26 promoter using a cell-free reconstitution system derived from fly embryos. Both DH sites were readily reconstituted from extract components. They were separated by a nucleosome which was less strictly positioned than its in vivo counterpart. The interactions of GAGA factor and heat shock factor with their binding sites in chromatin occurred in two modes. Their interaction with binding sites in the nucleosome-free regions did not require ATP. In the presence of ATP both factors interacted also with nucleosomal binding sites, causing nucleosome rearrangements and a refinement of nucleosome positions. While chromatin remodeling upon transcription factor interaction has previously been interpreted to involve nucleosome disruption, the data suggest energy-dependent nucleosome sliding as main principle of chromatin reorganization.

Animals↗
PubMed1995

Drosophila chromatin and transcription.

In Drosophila transcription is differentially repressed by various aspects of chromatin, thought to represent distinct structural levels: the nucleosome core particle, the linker histone H1 and as yet undefined higher order structures. Heterochromatin serves as a paradigm for the latter level of organization, but maintenance of silencing at homeotic gene loci may also fall into this class. Recently GAGA transcription factor (GAF) was shown to counteract chromatin repression at all levels. The various effects of GAF could be explained by its ability to rearrange nucleosomal positions. Chromatin remodelling by GAF and other factors in vitro require activities that maintain a highly dynamic state of chromatin.

Animals↗
PubMed1994

ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor.

Genetic control elements are usually situated in local regions of chromatin that are hypersensitive to structural probes such as DNase I. We have reconstructed the chromatin structure of the hsp70 promoter using an in vitro nucleosome assembly system. Binding of the GAGA transcription factor on existing nucleosomes leads to nucleosome disruption, DNase I hypersensitivity at the TATA box and heat-shock elements, and rearrangement of adjacent nucleosomes. ATP hydrolysis facilitates this process, suggesting that an energy-dependent pathway is involved in chromatin remodelling.

Adenosine Triphosphate↗
PubMed1994

Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin.

Chromatin reconstituted in an extract from preblastoderm Drosophila embryos represses transcription by RNA polymerase II. We have assembled regularly spaced nucleosomes on DNA attached to paramagnetic beads enabling the efficient purification of chromatin templates for transcription studies. We have used diagnostic salt extractions to establish that transcriptional repression of immobilized chromatin was largely due to nucleosome cores. When purified H1 was incorporated into chromatin, resulting in increased repeat lengths to 200-220 bp, the contribution of H1 to transcriptional repression was negligible. If more H1 was added no regularly spaced chromatin was obtained and only under these conditions was transcriptional inhibition by H1 apparent. We conclude that efficient repression of transcription by polymerase II in this system does not require the presence of histone H1.

Animals↗
PubMed1994

The establishment of active promoters in chromatin.

The organization of eukaryotic genomes as chromatin provides the framework within which regulated transcription occurs in the nucleus. The association of DNA with chromatin proteins required to package the genome into the nucleus is, in general, inhibitory to transcription, and therefore provides opportunities for regulated transcriptional activation. Granting access to the cis-acting elements in DNA, a prerequisite for any further action of the trans-acting factors involved, requires the establishment of local heterogeneity of chromatin and, in some cases, extensive remodeling of nucleosomal structures. Challenging problems relate to the establishment of this heterogeneity at the level of the single nucleosome and to the mechanisms that operate when nucleosomal arrays are reorganized. Recent developments indicate that chromatin reconstitution in cell-free systems allows the biochemical analysis of the interplay between transcription factors and chromatin components that brings about regulated transcription.

Animals↗
PubMed1994

Nonradioactive, solid-phase DNase I footprints analyzed on an A.L.F. DNA Sequencer.

Solid-phase DNase I footprinting provides a powerful tool for analyzing the sequence-specific interactions of DNA binding proteins. Classically this type of assay requires radioactively labeled DNA molecules. Substitution of the isotope by fluorescein labeling of the DNA fragments enables the analysis of footprint patterns on a standard automated laser fluorescent (A.L.F.) DNA Sequencer. The combination of solid-phase footprinting technology and fluorescence-based nonradioactive detection of fragments has unique advantages over established footprinting technologies.

DNA-Binding Proteins↗